孕激素上调poFUT1基因表达及促进胚胎增殖与黏附机制的研究

发布时间：2018-03-06 00:05

本文选题：孕激素　切入点：胚胎植入　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:孕激素(Progesterone,P)在胚胎发育成熟和着床过程中起重要作用,孕激素不足可导致胚胎发育不良,及妊娠终止引起流产。孕激素通过调节与着床有关分子的表达促进胚胎的增殖和黏附能力。多种病理和生理过程受到蛋白质糖基化的影响,其中包括了胚胎发育与着床过程。O-糖基化和N-糖基化是蛋白质糖基化的主要形式。蛋白质的O-岩藻糖基化受蛋白质O-岩藻糖基转移酶(po FUTs,protein-fucosyltransferases)的催化,而po FUTs在促进胚胎发育与着床过程中的作用未见报道。激活蛋白-1(AP-1)是一种转录因子,转录因子家族由Jun和Fos的细胞同源物组成,参与细胞增殖、分化、和侵袭过程和内分泌基因的调节。研究发现,AP-1(c-Jun/c-Fos)广泛表达于人胎盘中,活化的AP-1具有调控胎盘的形成和胎儿发育的作用。本研究拟探讨孕激素对蛋白质O-岩藻糖基化的调控作用及影响胚胎增殖和着床过程。研究结果表明JAR细胞经孕激素作用,增加AP-1(c-Jun/c-Fos)的磷酸化表达,上调poFUT1的基因表达,并促进胚胎细胞的黏附能力与增殖能力。实验有助于揭示岩藻糖在着床糖生殖生物学中的作用及其机制,为临床医学提供研究及诊断治疗的新思路。方法:1.利用ELISA和Western blot检测健康未孕、早孕和流产妇女血清中孕激素和poFUT1的蛋白表达水平。通过免疫荧光的方法验证人绒毛组织中poFUT1的定位和表达。2.利用Western blot,免疫荧光和Real-time PCR等方法,观察孕激素对胚胎滋养层细胞中poFUT1基因和蛋白表达水平的影响,以及孕激素与米非司酮联合应用对poFUT1基因和蛋白表达水平的影响。3.利用Western blot,免疫荧光和Real-time PCR等方法,观察孕激素影响poFUT1的表达及对细胞增殖黏附功能的改变。4.为了探讨孕激素是如何调控poFUT1表达,我们采用Western blot、EMSA和免疫荧光方法确定孕激素可激活转录因子AP-1的转录活性。5.利用染色质免疫沉淀技术检测转录因子AP-1直接作用在poFUT1的启动子区域,促进poFUT1转录,并且通过Western blot和Real-time PCR检测瞬时转染c-Jun/c-Fos siRNA干扰质粒和AP-1抑制剂(TIIA)处理后poFUT1基因和蛋白表达的变化。6.体外着床模型是在荧光显微镜下,观察和统计黏附率,检测了包括正常组、孕激素组、孕激素联合米非司酮组、转染c-Jun/c-Fos siRNA干扰质粒组,AP-1抑制剂(TIIA)组以及分别联合孕激素等9组的胚胎黏附率。并且利用Western blot、免疫荧光和CCK-8检测JAR细胞增殖能力。结果:1.收集临床样品包括健康未孕、正常早孕和先兆流产女性血清进行ELISA检测发现:正常早孕妇女血清中,孕激素和poFUT1的含量均高于先兆流产患者和健康未孕的女性;而先兆流产患者血清中,孕激素和poFUT1的含量比早孕妇女低。孕激素和poFUT1在血清中的表达变化呈正相关。通过免疫荧光确定人早孕绒毛中poFUT1的定位和表达。2.孕激素促进poFUT1的基因和蛋白水平的表达。经过不同浓度的孕激素和不同作用时间处理后,通过Real-time PCR和Western blot等方法检测发现,孕激素能显著促进JAR细胞poFUT1基因和蛋白的表达;Real-time PCR、Western blot和免疫荧光结果表明,联合应用米非司酮(RU486)可抑制孕激素的效用;瞬时转染poFUT1 siRNA后,poFUT1表达明显降低,联合孕激素处理后可回调poFUT1的表达。3.孕激素促进poFUT1的表达并调节JAR细胞的增殖与黏附的能力。Real-time PCR、Western blot和免疫荧光结果发现,干扰poFUT1的JAR细胞中合并应用孕激素可以恢复poFUT1的表达。通过Western blot、CCK-8和免疫荧光等方法分析发现孕激素调控poFUT1的表达影响JAR细胞的增殖和黏附能力。4.孕激素激活转录因子AP-1的转录活性。Western blot、EMSA和免疫荧光结果发现,孕激素(10-5mol/L)可上调转录因子AP-1(c-Jun/c-Fos)的表达水平,并促进其在核中的积累。5.AP-1直接影响靶基因poFUT1的转录,Real-time PCR、Western blot结果显示,与对照组相比,抑制了AP-1的转录激活能力可降低poFUT1的表达水平。通过CHIP实验进一步确定了AP-1直接作用在poFUT1启动子区域,引起下游靶基因poFUT1转录并调控其表达变化。6.孕激素通过激活转录因子AP-1上调poFUT1表达水平。孕激素可调控poFUT1的表达,poFUT1的表达变化可影响胚胎细胞增殖能力。通过Western blot、CCK-8和免疫荧光分析发现,与对照组相比,孕激素能提高JAR细胞的增殖能力;c-Jun/c-Fos siRNA能降低JAR细胞的增殖能力;联合孕激素处理可一定程度的回调由c-Jun/c-Fos siRNA引起JAR细胞增殖能力降低的现象;同时,孕激素也能够回调由AP-1抑制剂(TIIA)引起JAR细胞低的增殖能力;研究发现,孕激素可提高胚胎细胞的增殖能力。7.利用体外黏附模型,观察胚胎细胞的体外黏附率。孕激素可提高JAR细胞的黏附率;瞬时转染c-Jun/c-Fos siRNA干扰质粒可降低JAR细胞的黏附率;同时,孕激素能够恢复c-Jun/c-Fos siRNA预处理的JAR细胞的低黏附率;孕激素也能够恢复预处理AP-1抑制剂TIIA的JAR细胞低黏附率;进一步的研究发现孕激素可恢复poFUT1 siRNA导致的胚胎较低的黏附率。孕激素通过促进poFUT1的表达提高了胚胎的黏附能力。结论:1.正常早孕时期女性血清中孕激素和poFUT1的含量较先兆流产患者高,且呈正相关性。人绒毛组织上有poFUT1的表达。2.孕激素促进poFUT1基因与蛋白的表达水平。3.孕激素激活转录因子AP-1(c-Jun/c-Fos),并上调poFUT1的表达。4.孕激素促进胚胎细胞增殖能力。5.孕激素通过激活转录因子AP-1(c-Jun/c-Fos)的转录活性,进而调控靶基因poFUT1的转录,促进胚胎细胞与子宫内膜细胞的黏附。
[Abstract]:Background and purpose: progesterone (Progesterone, P) play an important role in the maturation of embryo implantation and process, progesterone deficiency can lead to embryonic dysplasia, and progesterone induced termination of pregnancy abortion. The ability to promote the proliferation and adhesion of embryo implantation and by regulating the expression of related molecules. A variety of physiological and pathological processes affected by protein glycosylation, including the process of embryo development and implantation of.O- glycosylation and N- glycosylation is the main form of glycosylated proteins. O- fucosylated proteins by protein O- fucosyltransferase (PO FUTs, protein-fucosyltransferases) and Po FUTs in catalysis, promote the process of embryo development and implantation. The role has not been reported. Activated protein -1 (AP-1) is a transcription factor family of transcription factors by Jun and Fos cellular homologues, involved in cell proliferation, differentiation, and invasion process and Regulating the secretion of genes. The study found that AP-1 (c-Jun/c-Fos) is widely expressed in human placenta, the activation of AP-1 can regulate the formation of the placenta and fetal development. This study intends to explore the role of progesterone on protein O- fucosylated regulation and proliferation and embryo implantation process. The results show that the JAR cells were treated with progestin. AP-1 (c-Jun/c-Fos), increased the expression of phosphorylation, upregulating the expression of poFUT1 gene, and to promote the adhesion and proliferation ability of embryonic cells. The results are helpful to reveal the fucose sugar in implantation in the reproductive biology effect and mechanism, and provides a new way to research the diagnosis and treatment for clinical medicine. Methods: using ELISA and 1. Western blot detection of the expression level of healthy non pregnant, pregnancy and abortion protein progesterone and poFUT1 in serum. The localization of poFUT1 by immunofluorescence test in human chorionic tissues And the expression of.2. by Western blot, immunofluorescence and Real-time PCR method to observe the effect of progesterone on the expression level of poFUT1 gene and protein in embryonic trophoblast cells, and the combined use of progesterone and mifepristone on poFUT1 gene and protein expression and the influence of.3. levels by Western blot, immunofluorescence and Real-time PCR methods, observe the effects of progesterone on poFUT1 the expression and change of.4. on cell proliferation adhesion function in order to investigate the progesterone is how to regulate the expression of poFUT1, we use Western blot EMSA and immunofluorescence method to determine the transcriptional activity of.5. progesterone can activate transcription factor AP-1 by chromatin immunoprecipitation assays of AP-1 transcription factor technology directly precipitated in the poFUT1 promoter region, promote poFUT1 transcription and through the Western blot and Real-time PCR detection of transient transfection of c-Jun/c-Fos siRNA plasmid and AP-1 interference suppression Preparation of (TIIA) changes of.6. implantation model in vitro poFUT1 gene and protein expression after treatment is under the fluorescence microscope, observation and statistics of the rate of adhesion was detected including normal group, progesterone group, progesterone and mifepristone group, transfection of c-Jun/c-Fos siRNA plasmid group, AP-1 inhibitor (TIIA) group and 9 group were combined with progesterone the adhesion rate of embryo and the use of Western. Blot, immunofluorescence and CCK-8 JAR detection ability of cell proliferation. Results: 1. collected clinical samples including healthy non pregnant women, normal pregnancy and threatened abortion of ELISA in serum were detected: the serum of normal pregnancy, progesterone and poFUT1 content were higher than that of threatened abortion patients and healthy non pregnant women the serum of patients with threatened abortion; however, the content of progesterone and poFUT1 than pregnant women. Low expression of progesterone and poFUT1 in serum were positively correlated with immune. Fluorescence to determine the position of poFUT1 in the villi and the expression of.2. and progesterone promotes the expression of poFUT1 gene and protein level. After different concentrations of progesterone and different time after treatment, detected by Real-time PCR and Western blot found that progesterone can significantly promote the expression of poFUT1 gene and protein in JAR cells; Real-time PCR, Western blot and immunofluorescence results show that the combined application of mifepristone (RU486) can inhibit the progesterone effect; transient transfection of poFUT1 siRNA, poFUT1 expression was significantly reduced after the treatment of combined progestin adjusting the expression of poFUT1.3. and progesterone can promote the expression of poFUT1 and JAR cell proliferation and adhesion regulating ability of.Real-time PCR, Western blot and immunofluorescence. The results showed that interfering the expression of poFUT1 JAR cells in combination with progesterone can restore poFUT1. By Western blot, CCK-8 and immune Fluorescence analysis found that progesterone regulates poFUT1 expression JAR cell proliferation and adhesion of.4. progesterone activated transcription factor AP-1 transcription activity of.Western blot EMSA, and immunofluorescence results showed that progesterone (10-5mol/L) can upregulate the transcription factor AP-1 (c-Jun/ c-Fos) expression levels, and promote its nuclear accumulation in.5.AP-1 effect of transcription of the target gene of poFUT1 Real-time PCR and Western blot results showed that compared with the control group, the inhibition of AP-1 transcriptional activation ability can reduce the expression level of poFUT1. A direct role for AP-1 in the promoter region of poFUT1 was determined by CHIP experiment further, causing downstream target gene poFUT1 transcription and regulation of the expression of.6. and progesterone through the activation of the transcription factor AP-1 up-regulated the expression level of poFUT1. Progesterone can regulate the expression of poFUT1, poFUT1 expression changes can affect embryonic cell proliferation. By Western blot, CCK-8 and immunofluorescence analysis showed that compared with the control group, progesterone can enhance the proliferation of JAR cells; c-Jun/c-Fos siRNA can reduce the proliferation of JAR cells in combination with progesterone treatment; callback to some extent decreases due to the proliferation ability of JAR cells by c-Jun/c-Fos siRNA phenomenon; at the same time, progesterone can also callback by AP-1 inhibitor (TIIA) JAR cells induced by low proliferation; the study found that progesterone can improve the adhesion in vitro model of embryonic.7. cells proliferation, embryonic cells in vitro. The adhesion rate of pregnant hormone can improve the adhesion rate of JAR cells; transient transfection of c-Jun/c-Fos siRNA plasmid can reduce the adhesion rate of JAR cells; meanwhile, progesterone can recovery of c-Jun/c-Fos siRNA pretreatment of JAR cells with low adhesion rate; progesterone can recover pretreatment of AP-1 inhibitor of TIIA JAR cells with low adhesion A further study found that the adhesion rate; progesterone can restore the poFUT1 siRNA leads to embryonic lower rate. Progesterone by promoting the expression of poFUT1 increased the adhesion ability of embryos. Conclusion: the content of progesterone and poFUT1 in serum of 1. normal pregnant women during the period of a threatened abortion were high, and there was a positive correlation between. Human chorionic tissues on poFUT1 the expression of.2. and progesterone promote poFUT1 gene and protein expression levels of.3. and progesterone activates the transcription factor AP-1 (c-Jun/c-Fos), and the increased expression of poFUT1.4. and progesterone promote embryo.5. cell proliferation ability of progesterone through activation of the transcription factor AP-1 (c-Jun/c-Fos) transcription activity, transcription and regulation of target gene poFUT1, promote adhesion of embryonic cells and endometrial cells.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714

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